System and workstation for the design, fabrication and assembly of bio-material constructs

ABSTRACT

A bioassembly system having a tissue/object modeling software component fully and seamlessly integrated with a robotic bioassembly workstation component for the computer-assisted design, fabrication and assembly of biological and non-biological constructs. The robotic bioassembly workstation includes a six-axis robot providing the capability for oblique-angle printing, printing by non-sequential planar layering, and printing on print substrates having variable surface topographies, enabling fabrication of more complex bio-constructs including tissues, organs and vascular trees.

PRIORITY CLAIM

This application is a continuation of U.S. Non-Provisional applicationSer. No. 15/873,453, filed on Jan. 17, 2018, which is a continuation ofU.S. Non-Provisional application Ser. No. 14/511,693, filed on Oct. 10,2014, which claims priority to U.S. Provisional Application No.61/889,856, filed on Oct. 11, 2013, and U.S. Provisional Application No.62/016,815 filed on Jun. 25, 2014, the entire disclosures of which areincorporated herein by this reference.

TECHNICAL FIELD

The disclosure relates to computer-assisted design of tissue structuremodels, robotic fabrication and bioassembly, and a workstation for thedesign, fabrication and assembly of complex biological constructs. Thebioassembly system enables users to design/model, fabricate and/oranalyze complex tissue structures facilitated by novel Tissue StructureInformation Modeling (TSIM) software.

BACKGROUND

Tissue engineering via 3-D biomaterial dispenser-based precisiondeposition is a fast-evolving technology that has gone from provocativescience fiction to the realization of 3-D “bioprinted” functional organslivers in just a little over a decade. For simplicity, as used herein“bio-printing” refers broadly to any biomaterial dispensing technologyutilizing three-dimensional, precise deposition of biomaterials viamethodology that is compatible with an automated, computer-aided,three-dimensional prototyping device (a bioprinter). Computer assisteddesign (CAD)-facilitated 3-D bioprinters are now available as retailproducts, and companies engaged in commercializing bioprinters andbio-printed products are publicly traded on the New York Stock Exchange.The rapid growth in the 3-D tissue engineering industry is in large partclue to a demand for transplantable organs and organ repair tissues thatis increasing at a faster rate than the supply. Hence, the prospect forurgent time-frame, large volume fabrication of synthetic biologicalconstructs, including functional tissues and organs, has wide-spreadappeal and has achieved significant private and government resourcecommitment.

Bioprinting and bioprinters have advanced significantly in recent years;however a stand-alone bioprinter still has very little usefulfunctionality. End-users must often develop their own software and setup suitable workstations—tasks requiring expertise incomputer-assisted-design, electronics and related materials engineering,as well as in the relevant biological science. Hence, without resourcesfor and access to a team of experts, designing and bioprintingbiological constructs such as tissues and organs remains the province oflarge well-funded research organizations and entities.

Conventional tissue engineering systems based on bioprintingtechnologies also typically require large work spaces because design,printing and assembly are typically effectuated on different platforms.

State-of-the-art bioprinters offer a wide variety of functionality, inparticular at the dispensing end, where consumers may choose from singleto multi nozzle print heads and from a wide range of biomaterialdispensing mechanisms. Contact-based deposition techniques such as softlithography and non-contact based deposition techniques such aspressure-actuated ink jetting and laser-guided direct writing, have allbeen exploited in bioprinter design. Most bioprinters, however, rely onmovement of the print head along three axes, which achieves precisedeposition in two dimensional planar coordinates, but which limitsbuilding complex tissue and organ constructs to a layer-by-layerprotocol, resulting in build support complications and other buildchallenges in multi-tissue constructs.

There remains a need for a technologically comprehensive tissue designand fabrication workstation fully integrated with tissue modeling andoperational software to provide user-friendly functionality forCAD-assisted tissue engineering, and a need for workstation designswhich achieve modeling, fabrication and assembly in a more compact workspace. Further, there remains a need for bioprinter designs that providegreater flexibility in build protocols.

SUMMARY

Accordingly, embodiments of the invention provide an intuitive,user-friendly tissue structure design and fabrication system, referredto herein as a bioassembly system, facilitated by software thatseamlessly integrates the design and fabrication modalities. Thebioassembly system comprises two major components: (i) software for“Tissue Structure Information Modeling” (TSIM) and (ii) hardware forstaging, printing and assembling No-constructs, referred to as the“Robotic Bioassembly Workstation” (RBW). The RBW provides a compact, andin some embodiments, a mobile workspace, in which print/fabrication andassembly may take place on the same platform. The robotic aspect iscapable of switching between different modalities including staging,printing and assembling. Greater build versatility is achieved byutilization of a robotic arm capable of movement along at least sixdifferent axes, thus providing capability of dispensing biomaterial bynon-sequential planar layering and by providing capability of dispensingon 3-D surfaces of variable surface topographies.

One embodiment of the invention provides a bioassembly system comprisinga tissue modeling component and a robotic bioassembly workstationcomponent. The tissue modeling component comprises a user interface, atleast one suite of tools for performing an object operation selectedfrom the operation categories of creating, editing, modeling,transforming, image property modulating, sketching, print supporting,simulating, material testing and combinations thereof, a materialdatabase, and software executable by a machine to facilitate a methodfor designing a volumetric model of a biological construct at the userinterface.

The tissue modeling component is operationally linked to a roboticbioassembly workstation component and the method comprises: adding atleast one object to an object modeling environment at the TSIM-userinterface, wherein adding comprises selecting, creating, importing andcombinations thereof, and further wherein each added object may beassociated with an object list comprising material and/or environmentalbuild parameters; performing one or more operations on the one or moreobjects in the modeling environment to render a desired volumetricmodel; transmitting the rendered volumetric model to the roboticbioassembly workstation with a print and/or assembly command; andprinting and/or assembling the modeled object as a bioconstruct.

According to some embodiments, a simulation or materials test may beconducted prior to effectuating the printing/fabricating command.

Another embodiment is directed to a robotic biomaterial dispensingapparatus comprising a robotic arm and a robotic arm end effector. Theend effector is configured to grip and secure a dispensing syringe andthe robotic arm provides movement of the syringe along at least sixaxes.

Other embodiments are directed to the bio-constructs designed,fabricated and assembled utilizing the bioassembly system and accordingto the inventive methods.

These and other embodiments will be more clearly understood andappreciated by reference to the figures and detailed disclosure setforth below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 . Depicts an illustrative robotic bioassembly workstation (RBW).

FIG. 2 . Depicts an illustrative RBW robotic arm effector gripping asyringe barrel loaded with biomaterial and positioning the syringe forprinting over printing stage.

FIG. 3 . Depicts exemplary printing of biomaterial via direct-writedispensing from a biomaterial-loaded syringe,

FIG. 4 . 4A depicts an exemplary object modeling environment at the TSIMuser interface; 4B depicts an exemplary TSIM operations tool panel inthe object modeling environment.

FIG. 5 . 5A depicts an exemplary volumetric object menu showing displayof an object list once an object is selected; 5B depicts an exemplarymodeling environment displaying an object list materials selection.

FIG. 6 . Depicts a specific modeling environment screen illustrating anexemplary transformation operation panel.

FIG. 7 . Depicts specific modeling environment screens illustratingcreating a box; 7A depicts setting the base of a box; 7B depicts settingthe height of the box; 7C depicts a final volumetric box.

FIG. 8 . Depicts specific modeling environment screens illustratingcreating a sphere; 8A shows setting the diameter of a sphere; 8B shows afinal volumetric sphere.

FIG. 9 . Depicts specific modeling environment screens illustratingcreating a cylinder; 9A shows setting the base of a cylinder; 9B showssetting the height of the cylinder; 9C shows a final volumetriccylinder.

FIG. 10 . Depicts specific modeling environment screens illustratingcreating a pyramid; 10A shows setting the base of a pyramid; 10B showssetting a height of the pyramid; 10C shows a final volumetric pyramid.

FIG. 11 . Depicts specific modeling environment screens illustrating asimple addition operation; 11A select first object; 11B select secondobject; 11C click addition operation icon to form unified object.

FIG. 12 . Depicts specific modeling environment screens illustrating asimple difference operation; 12A select active object first; 12B selectinactive object next; 12C click difference operation icon to leaveportion of active object that does not intersect with inactive object.

FIG. 13 . Depicts specific modeling environment screens illustrating anintersection operation; 13A select active object as first object; 13Bselect second object; 13C click intersection operation icon to leaveportion of active object that intersects with first and second objects,

FIG. 14 . Depicts specific modeling environment screens illustrating analignment operation; 14A select a point on desired face of first object;14B select point on desired face of second object; 14C click alignoperation icon and first object will move such that points on selectedfaces align.

FIG. 15 . Depicts specific modeling environment screens illustrating twomore TSIM operations; 15A dragging; and 15B measuring.

FIG. 16 . Depicts a specific modeling environment screen illustratingadjusting the volume of a DICOM image imported into the object modelingenvironment.

FIG. 17 . Depicts a specific modeling environment screen illustrating anextrusion operation to convert a sketch into a volumetric model.

FIG. 18 . Depicts specific modeling environment screens illustratingcreation of new volumetric objects by lofting contours; 18A loftingcontours of a solid T-form; 18B sketches setting base and top surfacesto create a volumetric object; and 18C lofting contours of thevolumetric object to create a new object.

FIG. 19 , Depicts a specific modeling environment screen illustratingthe “create a tube” operation; 19A depicts selecting a radius for anunbounded sketch in the modeling environment; and 19B click the “createa tube” operation icon to form a volumetric tube.

FIG. 20 . Depicts a specific modeling environment screen for creation ofa vascular tree and an exemplary vascular tree,

FIG. 21 . Depicts a very specific exemplary print screen at the TSIM-UI.

DETAILED DESCRIPTION

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. All publications, patentapplications, patents, and other references mentioned herein areincorporated by reference in their entirety.

As used in this specification and the appended claims, the singularforms “a,” “an,” and “the” include plural references unless the contextclearly dictates otherwise, Any reference to “or” herein is intended toencompass “and/or” unless otherwise stated.

As used herein, “biomaterial” means a liquid, semi-solid, or solidcomposition comprising a plurality of cells, cell solutions, cellaggregates, multicellular forms or tissues, and in all cases may includesupport material such as gels, hydrogels, alginate or non-cellularmaterials that provide specific biomechanical properties that enablebiomaterial printing.

As used herein, “cartridge” means any object that is capable ofreceiving (and holding) a biomaterial and/or a support material and usedinterchangeably with “syringe barrel.”

A bioassembly system comprising a tissue modeling component and arobotic bioassembly workstation component is disclosed and detailedherein. The bioassembly system is an integrated solution for tissuestructure modeling, fabrication and assembly comprising a softwarecomponent referred to herein as Tissue Structure Information Modeling orTSIM, and a hardware component referred to herein as a RoboticsBioAssembly Workstation, or RBW. TSIM permits clinicians and scientiststo design, visualize, simulate, and analyze three dimensional (3-D)computer models of complex biological constructs, including tissuestructures created from traditional sources of medical imagingtechnology. TSIM provides a computer-assisted-design (CAD) platform thatis particularly suited for end-users without specific expertise inconventional CAD software.

Generally, TSIM comprises software and a user interface comprising anobject modeling environment. TSIM comprises several suites of tools forperforming one or more object operations. Object modeling tools includebut are not limited to tool suites for creating, editing, modeling,transforming, image property modulating, sketching, print supporting,simulating, material testing and combinations thereof. In someembodiments a materials database is provided, and in specificembodiments objects stored in an object list are associated withspecific materials and material use parameters. FIG. 4B depicts anexemplary object list displayed in association with a selected object.The TSIM software is executable by a machine to facilitate methods fordesigning volumetric models of biological constructs at the TSIM-userinterface in what is referred to herein as an object modelingenvironment.

TSIM is operationally linked to the robotic bioassembly workstationcomponent such that once a user has completed a modeling task, the usermay send a print command (see FIG. 21 for an exemplary print commandscreen at the TSIM-UI) to the RBW to initiateprinting/fabrication/assembly.

For purposes of the following disclosure, reference to a particularExample and/or Figure is made to provide additional illustration/detailand explanation of the functionality/operation.

When integrated in a work flow, the bioassembly system designs,fabricates and assembles complex three-dimensional biomaterialconstructs, including but not limited to cellular systems, tissues,organs, and implantable medical devices and jigs. The RIM aspectcomprises a material storage unit including a syringe holder andmultiple cartridges (syringe barrels) each with a set of syringes, and arobotic arm component with an end effector capable of gripping, holding,and connecting specialized syringe, and a dispenser. According tospecific embodiments the bioassembly system ultimately achievesCAD-based extrusion dispensing of biomaterials. Suitable materialsinclude but are not limited to biomaterials such as cells, biosupportmaterials such as gels, and non-biological materials, for example in thedesign and fabrication of implantable jigs. Combinations ofbiomaterials, biosupport materials and non-biological materials may beutilized in the same fabrication.

As used herein, “dispensing of biomaterials” may be effectuated by anybioprinting technique including but not limited to inkjet,extrusion/microextrusion, and laser-assisted printing. Thermal inkjetprinters electrically heat the printhead to produce air-pressure pulsesthat force droplets from the nozzle, while acoustic printers use pulsesformed by piezoelectric or ultrasound pressure. Extrusion printerstypically rely on pneumatic or mechanical (piston or screw) dispensingmechanisms to extrude continuous beads or filaments of biomaterial (orbiomaterial). Laser-assisted printers use lasers focused on an absorbingsubstrate to generate pressures that propel cell-containing materialsonto the substrate. According to preferred embodiments, the roboticallycontrolled bioprinting of the RBW comprises extrusion dispensing onto asubstrate.

According to particular embodiments, a user at the TSIM-UI may “add” oneor more objects to an object modeling environment. An exemplary TSIM-UIscreen showing an object modeling environment along with an operationstool panel is set forth in FIGS. 4A and 4B, respectively. Adding may beeffectuated by different functional capabilities. An object may be addedby selecting from a panel linked to a stored database of pre-existingobjects (FIG. 5A). In specific embodiments the panel comprises one ormore of a cube, a cylinder, a sphere and a pyramid as basic shapes. Theshapes may be edited and/or transformed and stored as new objects on thepanel for future applications. In specific embodiments, other objectsmay be added to the stored panel and it is contemplated that a user willadd/create and import objects to yield a customized menu/panel of storedobjects from which to select for addition to the object modelingenvironment.

In other embodiments, objects may be created directly in the objectmodeling environment. As illustrated in Example 1, by using the basiccube/box (FIG. 7 ), sphere (FIG. 8 ), cylinder (FIG. 9 ) and/or pyramid(FIG. 10 ) operations, a user may create basic shapes/objects and thenmanipulate, modify, edit, transform, add, intersect, e.g. the objects toderive a number of novel volumetric objects. The term “volumetric” isused herein to mean solid, three-dimensional forms.

In specific embodiments, an object may be created in the modelingenvironment by sketching and then performing operations on the sketch.Basic sketching functionality is illustrated in Example 5. A user mayalso sketch a 2-dimensional (2-D) bounded construct and perform anextrude operation (Example 6 and FIG. 17 ) in at least one direction toform a novel model volumetric object. A user may sketch boundedconstructs on multiple planes in xyz space and connect particularboundaries to form novel objects, and a user may loft one or more of thecontours of any object or sketch to varying degrees to further createnew volumetric objects (Example 7 and FIG. 18 ).

One specific embodiment exemplified in FIG. 19 and Example 9 provides atube operation whereby a user creates a curve in two orthree-dimensional space, selects a radius, and performs a tube operationto yield a tube. Tubes, like all other object models, may be edited,transformed, modified, combined with other objects, and the like.According to another specific embodiment, a user may create a vasculartree model by performing a vascular tree operation by setting aparameter profile as exemplified in Table 2 (FIG. 20 and Example 8).Non-limiting examples of input parameters for formation of a modelvascular tree comprise starting diameter, order length, ratio, number oforders, and ending diameter. A vascular tree may be fabricated on aprint substrate having variable surface topographies to provide a highlyintricate and embedded vascular construct for fabrication into morecomplex bio-constructs such as tissues and organs.

According to other embodiments, an object may be added to the modelingenvironment by importing an object from an external file. Users may openexisting model files created in external programs and import themdirectly through TSIM. Exemplary importable file formats include STL,NIfTI and DICOM. Specific embodiments illustrating importing in each ofthese file formats are set forth in Example 3. External files derivedfrom any medical imaging technology may be imported, although in certainspecific embodiments, adaptation or conversion of the file may benecessary. Non-limiting examples of medical imaging technology fromwhich importable images may be derived include magnetic resonanceimaging, X-ray radiography, medical ultrasound, endoscopy, elastography,tactile imaging, thermography, medical photography and positron emissiontomography.

Data generated from medical imaging technology is imported to TSIM,analyzed, and used to generate 3-D models or model scaffolds and/ortissue constructs customized to be patient-specific. In certainembodiments, tissues, organs, medical devices and medical jigs may bemodeled and fabricated clinically in situ based on specific needs of apatient.

The bioassembly system includes a TSIM user interface which permits userinput to guide the model-generation process. 3-D models may be combinedwith pre-loaded “tissue engineering recipes” containing necessarymaterial and/or cell types, assembly parameters, and an assemblyinstruction sequences. TSIM may also include functionality forsimulation testing. The RBW may further comprise an environmentalsensing system including visualization capability, and in someembodiments is capable of providing verification feedback to a userduring the assembly process. In some cases assembly may includeintegration of biological with non-biological constructs.

The RBW allows users to sub-assemble and assemble complex tissuestructures, and to assemble tissue constructs into higher orderbiological constructs including organs. According to some embodiments,material cartridges/barrels are loaded manually into the RBW by theuser, and are placed in the syringe barrel holders. The RBWuser-interface may assist the user throughout the process of loading thematerial cartridges. In specific embodiments the RBW comprises aplurality of syringe barrels containing different materials that can bechanged automatically or manually. Other than space constraints, thereis no limit to the number of barrels which may be loaded into the RBWfor specific uses. In very specific embodiments the robotic workstationcomprises between 2 and 12 syringe barrels. In even more specificembodiments the robotic workstation comprises 12 syringe barrels.

The RBW is capable of conducting a change in tooling using aninterlocking mechanism of the barrel adapter and an interchangeablecomponent. A material storage system stores the interchangeablecomponents in a fixed orientation when not in use. A gripper temporarilyimmobilizes the interchangeable component relative to the gripper, whilea robotic joint (J6) performs a twisting motion of the barrel adapter tolock or unlock the mechanism. When in the locked position, the tool canbe used as needed.

To load material cartridges for printing, the robot moves to theappropriate storage rack location for pickup. After aligning with thesyringe position, the robot moves the adapter head of the end effectordirectly onto the top of the syringe barrel. The robot gripper engagesand raises the syringe barrel out of the holder. Next, the robot's J6motor locks the syringe barrel into the adapter head to prepare forassembly, after which the gripper disengages and the robot returns intothe home position.

Once the appropriate material cartridge has been prepared, loaded andpositioned for printing, the material is dispensed onto the RBW printstage based on the print/assembly path specified from TSIM.

Depending on the materials needed to produce a complex tissue structure,the robot swaps material cartridges as needed. In order to periodicallyclean the nozzles and ensure quality prints, the robot may migrate overto a cleaning surface and clean the needle tip. A dispensing syringe maybe fitted with needles of varying sizes and lengths, according toparticular project demands.

The RBW is an integrated workstation comprised of a multi-axis robot andcontroller, integrated cell and material cartridge containment anddispensing aspect, an environmental control aspect and intelligentvisual aspect, configured to provide a compact convenient table-topworkstation for bioconstruct assembly needs.

TSIM and the RBW operate together to provide a comprehensive integratedsoftware and hardware technology platform including medical imaginganalysis, biological construct modeling, physical simulation,fabrication and assembly.

According to one exemplary embodiment, TSIM is used to effectuateimporting patient-specific tissue structures and combining them into a3-D biological computer models with corresponding Tissue EngineeringRecipes (e.g., a heart valve, ear lobe, etc.) that contain bill ofmaterial and/or cell types, assembly specifications (e.g., environmentalcontrol) and assembly sequence (e.g., apply cell type I and thenintegrate a vascular network). In this manner, a properly engineeredtissue structure can be applied to specific patient biometrics toachieve a solution that is viable and practical for medicalreplacement/implantation utilities or, for example, laboratory testingof medicines and medical devices.

TSIM includes the capability to simulate a wide range of real worldvariables to analyze resulting outcomes. Simulation modules include, butare not limited to, material viscosity and assembled shape deformation,tissue structure analysis, vascular fluid dynamics, and the like.

The RBW is integrated seamlessly with TSIM to allow a user to obtain afully assembled bioconstruct or physical tissue structure of the 3-Dmodels developed within the software program. The multi-axis robotreceives assembly steps and information from the TSIM software thatincludes material types and appropriate environmental conditions (e.g.,temperature, UV light, humidity, etc.). The robotic visual systemprovides real time feedback to both the user and TSIM to intelligentlydetect assembly quality and for verification of design specifications.

In some embodiments, an automated materials/configuration testingprotocol is provided prior to fabrication. In certain embodiments theuser configures each syringe barrel or cartridge to provide uniquedispensing characteristics for each object or object feature printed.Prior to actual fabrication, test applications may be effectuatedwhereby a configuration may be adapted through observation or automaticindication via feedback to TSIM of the quality of printing. A testpattern may include, for example, an appropriate two-dimensional orthree-dimensional pattern to test and tune the print quality such asstart/stop/delay, line width, speed, pressure, and acceleration. Inspecific embodiments a user is able to stop a test print at any time,for example upon real-time observation or indication of an error. Oncefine-tuned, final configurations may be saved and transferred into TSIMfor future applications.

The RBW comprises a closed-loop cartridge dispensing system,“Cartridges” may be any material containment and dispensing article andmay be custom-engineered or purchased and contain specific materialincluding specific cell types, biological or non-biological structuralsupport material, and other materials as appropriate/desired forparticular assembly applications. A cartridge may also be referred toherein as a syringe barrel. The RBW permits loading or retrieval of aspecific material/cell cartridge that corresponds to what is specifiedin the TSIM-generated 3-D modeling protocol. When a TSIM 3-D model isready to be “printed,” the user loads the cartridges containing thematerial/cell types specified in the model into the robotic workstation.

According to specific embodiments user-error is substantially controlledthrough the workstation visualization and sensing system. For example,the workstation robot may scan a bar code affixed on the cartridge toverify and validate vital information such as material type, lot number,date, temperature etc. Laser displacement sensors may be used to ensurethat syringe barrels are properly seated and secured in the materialstorage unit.

Once verification has been completed, the robot employs a speciallydesigned end effector to grip and secure the cartridge and move to theprint/assembly stage within the workstation. End effectors may comprisethe capability to load multiple cartridges at one time to enhanceassembly time. The workstation robot then executes the assemblyinstructions and steps that it receives from TSIM.

Biological models are created with TSIM using different workflows.According to a first workflow, users can choose to build a model byimporting medical images and then performing operations on the model toshape and refine the final 3-D model to be used for printing. A modelmay be refined further by defining objects using the acquired 2-D imageset in an Advanced User 2-D Image Editor module.

According to a second workflow, users create models using basic shapes.In one aspect of the second workflow, a basic set of geometric shapes isavailable to users (e.g., cube, cylinder, sphere, pyramid), all of whichcan be selected and placed directly into the object modeling environmentand combined, stretched and deformed to meet specific application needs.In addition, manipulation commands such as *Difference, *Intersect, and*Union are available for the user to create a desired shape.

Once created, users may run simulations on a model to determine whetheror not the constructed output will be structurally sound after printing.After simulations confirm the structural integrity of a printedstructure, the user selects the Print command. Once activated, the Printcommand sends information to the RBW for object fabrication andassembly.

In very specific embodiments, TSIM utilizes Insight Toolkit (ITK), a C++library, in order to perform data registration and segmentation. Incertain embodiments, data used by TSIM may be found in digitally sampledrepresentations of tissue (i.e., images acquired from medicalinstrumentation such as CT and MRI machines), Segmentation is theprocess of identifying and classifying the data found in digitallysampled representations. Registration is the task of aligning ordeveloping correspondences between data sets. For example, a CT scan canbe aligned with an MRI scan in order to combine the informationcontained in both representations. ITK will allow TSIM to consumemedical data and then combine different data sets to provide the mostcomplete representation of a tissue structure to the user.

(ITK was originally developed by the US National Library of Medicine. Aconsortium was put together to manage the project, called the InsightSoftware Consortium. ITK is licensed under the Apache 2.0 license.)

Data Visualization and Modeling

In specific embodiments, TSIM utilizes Visualization Toolkit (VTK) forimage processing, modeling, and volume rendering. This toolkit easilyinterfaces with ITK, allowing images analyzed using ITK to be modeled in3-D. VTK, like ITK, is a C++ library. Thus, users are able to importimages into TSIM (powered by ITK), edit imported images, and visualizeconstructed 3-D models of tissue structure(s) (powered by VTK). (VTK islicensed under the BSD (3-Clause) License, which allows the commercialuse, modification, distribution, and sublicensing of the code.)

Medical Image File Format Support.

In very specific embodiments, TSIM supports a variety of standardimaging formats including DICOM, NIfTI, and the FLUOVIEW FV1000 Version2 OIF file format for importing medical imaging data and associatedmetadata. The DICOM file format is the Digital Imaging andCommunications in Medicine standard for handling, storing, printing, andtransmitting information in medical imaging. It includes a file formatdefinition with a set of tags that contain information such as pixelformat, modality, and patient information. Images stored in the DICOMformat can be stored as a series of DICOM files in which each contains asingle image plane. The NIfTI file format stems from the NeuroimagingInformatics Technology Initiative, a working group sponsored by theNational Institute of Health. The file includes a header and data. Theheader includes such information as the pixel format and depth, pixelspacing, and X, Y, Z, T image dimensions. If multiple image planes aresaved, NIfTI files store the data as one all-encompassing file. TheFLUOVIEW FV1000 Version 2 OIF file format is a specific confocalmicroscopy format that contains header information within the OIF(Original Imaging Format) file and within the individual PTY files. Thisformat also saves Tagged Image File Format (TIFF) image files and thePTY files reference the TIFF images and contain the pixel data anddimensioning to be used when reconstructing the image stack. This fileformat is capable of storing multiple channels of data. In certainembodiments TIM uses the first channel.

Imported Image Quality.

TSIM provides users the option to import medical imaging data at full orlower resolution. The user is presented with a quality sliding bar, thatwhen adjusted either increases or decreases the resolution of a previewimage. For comparison, the original full quality image may also bepreviewed. Below the preview images, a thumbnail slider may be availableso that the user can view the effects of the quality changes on multipleportions of the image set. The added value of this functionality is thatit will free up RAM for additional computing power that can be used forother TSIM operations.

3-D Model Editor—Loaded Volume

After importing an image set, a set of preview images is presented tothe user, each of which has a different preset filter applied. The userselects the most desirable image according to the needs of theapplication. The user will then be presented with another set of previewimages that have the first filter they selected applied but now with anadditional filter applied. The user will then pick the most desirableimage, and this process will continue for multiple, for example 3, totaliterations. After the process is over, a 3-D model is generated usingthe preset filters indirectly chosen by the user.

This 3-D model can then be edited to show the data that the user is mostinterested in capturing. Initial 3-D model editing may be done usingTSIM tools. Tools available in TSIM to the user for creating, editingand transforming objects in the object modeling environment are setforth below.

Spline Tool: This tool will allow the user to define splines within the3-D model. These splines may be used as reference lines. A user drawssplines to define the boundary of objects within the 3-D model and theseare used in the generation of the final solid 3-D model. If the modelhas been constructed using imaging data, while utilizing the spline toolthe user will be able to use the mouse scroll wheel to scroll up anddown the model through the 2-D image stack as can be seen in the heartmodel of Figures x. The user is able to scroll to a desired image in thestack and snap their splines to that image plane in order to betterdefine tissue structures.

Region of Interest Tool: This tool can be used by the user to define aregion of interest within the 3-D model by creating a closed loop (endsare connected) free form spline around a desired portion of the 3-Dmodel. The user must orient the model into the desired view beforemaking the spline. After the region has been defined by the user, thespline will extend infinitely in the z direction. The 3-D model willthen update and exclude all data that wasn't included in the region ofinterest (Figure x).

Eraser Tool: This tool turns the user's mouse cursor into eraser in theshape of a sphere within the 3-D rendering window. After selecting thetool, the user can erase image data by clicking and holding the leftmouse button. Additionally, the eraser size can be changed to facilitatethe erasure of smaller or larger features. The user can then switch thetool off and the 3-D model will update automatically.

Cube Tool: This tool permits users to create cubes in model space. Onceselected, a standard cube may be generated in the model space that canbe resized and moved using manipulators located on the cube. Inaddition, input will be available for coordinate positioning, forexample, X, Y, and Z positioning, and for scale of the object.

Cylinder Tool: This tool allows users to create cylinders in modelspace. Once selected, a standard cylinder will be generated in the modelspace that can be resized and moved using manipulators located on thecylinder. In addition, input will be available for coordinatepositioning, for example, X, Y, and Z positioning, and for scale of theobject.

Sphere Tool: This tool allows users to create spheres in model space.Once selected, a standard sphere will be generated in the model spacethat can be resized and moved using manipulators located on the sphere.In addition, input will be available for coordinate positioning, forexample, X, Y, and Z positioning, and for scale of the object.

Exemplary 3-D Modeling Tools

Pan—This tool is used to shift the viewing angle of a modeling spaceleft, right, up or down.

Rotate—This tool is used to rotate the 3D model about, for example, a3-dimensional axes (X, Y, Z).

Zoom—This tool is used to magnify or de-magnify a model as it ispresented on the computer screen.

Intersection—This tool is used to define a region boundary based on theintersection of two splines/objects.

Difference—This tool is used to define a region boundary based on thesubtraction of an actively selected object from an inactive object.

Union—This tool is used to define a region boundary based on theaddition of two object volumes (FIG. 9 ).

Object Map—Defined boundaries may be tracked and defined objects may bepresented to the user in a color coded object map. This allows the userto easily visualize the individual components of their 3-D model.

Annotation—A text annotation tool may be added to the application. Thetext annotation tool permits a user to add text notes to a selected edgeor filled region. An option exists added for leader lines to point outthe area of interest for a note that is added.

Measurement—This tool allows users to draw point to point measurementlines on both a 2-D image slice and within a 3-D model. This may beuseful, for example, when the user is dimensioning the 3-D model forprinting. In addition, a scale bar and a reference object (such as acoffee cup) may be supplied in the 3-D modeling environment.

Advanced User 2-D Image Editor User Interface—This interface presentsdata to a user as a sequence of grayscale two-dimensional slices, withintensity determined by the density, of the tissue. Structures areidentified and isolated using the varying intensity as a guide.Isolation of a structure is accomplished by creating free-form boundedregions on each slice, which are then connected together between theslices to calculate an isolated volume.

TSIM also provides different tools for controlling image properties,such as brightness and contrast, and for image editing (e.g.,annotations and object definitions). A loaded image set will bedisplayed below the image workspace using scrollable image previews.Within the image workspace, a 3-D rendering of the image set will bedisplayed with three image views that can be collapsed. Exemplary viewsinclude axial, sagittal and coronal views which, for example, may belocated in the upper left corner, bottom left corner, and bottom middleof the screen when restored.

Exemplary Image Property Control Tools:

Brightness, for increasing or decreasing the tonal values of the image

Contrast, for expanding or shrinking the overall range of tonal valuesin the image.

Window Midpoint which may be represented as a dial that can be used toadjust the midpoint of the density mapping into grayscale for the imagebeing viewed.

Window Range for expanding or shrinking the visible density range of theimage being viewed.

Filters numerous filter presets may be available for the user to applyto the image set.

2D Rectangle Inclusion Tool permits the user to define a rectangularregion of interest within a 2D image. After the region has been definedby the user, the 3D model will then update and exclude all data that wasnot included in the region of interest.

Image Reset Tool reverts the image processing to the original 2D imagelevels.

2D Boundary Definition Tools

Point Spline—This tool is used to define a region boundary from a closedspline created from a series of control points. Each input within theimage area will add a new control point. If desired, TSIM willautomatically adjust control points to better fit the boundary to nearbydetected edges. After the boundary has been completed, the user maydirectly adjust the position and parameters of any control point, aswell as add or remove control points. Once the user is satisfied, theycan confirm the boundary and a region object will be created. The PointSpline Tool acts as an editor if the user would like to make changes tothese region objects in the future.

Free Form—This tool allows the user to create a spline from a free-formsketch. After creating a sketch, the software fits a spline to thesketch. The user may draw over this spline to refine the shape. Once theuser is satisfied, they can confirm the boundary and a region objectwill be created. The Point Spline Tool acts as an editor if the userwould like to make changes to these region objects in the future.

Duplicate—This tool copies bounded regions from one image slice toanother. This tool may be used to avoid the effort of creating regionswhen the differences between image slices are minimal. After duplicatingregions, the user may use the Point Spline Tool to make any necessarycorrections.

The TSIM User Interface (II)

The TSIM UI displays the planned print path of the 3-D model and thecurrent print head location along the path in real time. In addition,the printer's current state regarding the parameters of active nozzle,pressure, and speed is displayed along with an active print duration. Achart showing the amount of material used by the printer may bedisplayed on the right-hand side of the UI. Below the print path, ascrollable timeline may be generated that allows the user to track theprogression of the printed material throughout the print cycle. Dialogboxes may pop up if an error is encountered in the printing process. Forexample, if the printer runs out of material in a cartridge, a dialogbox will pop up on screen that says, “Fill Reservoir 2 with Gel 2.”

A TSIM object modeling screen provides a 3-D environment fordesigning/creating models, or for importing existing models for thepurpose of printing/fabricating via the robotic workstation. Complexmodels may be created using a basic set of geometric shapes. Anillustrative provided set of geometric objects comprises box, cylinder,sphere and pyramid shapes. Manipulation tools include, but are notlimited to providing functionality of combining, aligning, sketching,volume-rendering, surface-rendering, and the like. Models may be openedfor solid modeling in a variety of standard imaging formats, includingfor example STL, DICOM and NIfTI formats. An Exemplary TSIM operationspanel is depicted in Figure X.

Each object added to the object modeling environment is associated withan object property list such that the object property list is displayedupon adding a selected object to the modeling environment, and acomposite object property list is displayed comprising an objectedproperty list for each object added to the modeling environment. FigureX depicts an exemplary modeling environment including four selectedobjects and a corresponding composite object property list. The objectsare selected from cube, cylinder, sphere and pyramid. Selected orcreated objects may be given unique identifiers by the user.

Key Frame Automatic Bounding Support. TSIM includes a Key FrameAutomatic Bounding Protocol in which intermediate region boundaries willbe extrapolated between two non-adjacent image slices. Typically, thesetwo images will provide the clearest views of objects within the imagesthat the user wants to define. The user will select two non-adjacentimage slices as key frames and execute the Extrapolate command. Thiscommand tells the program to extrapolate those defined features throughthe images in between the defined key frames using the edge detectionalgorithm.

Extrusion Material Assignment Tool. This tool allows the user to specifya set extrusion material for defined objects in the 3-D model or todefine regions of the 3-D model as consisting of a certain material. Forthe first case, the user selects the tool, the extrusion material, andthen the desired object(s). For the second case, the user may defineareas using existing splines or by drawing a spline and then selectingthe tool. The user may then either specify the program to define amaterial region from a spline using a specified thickness or byextending the region to another spline.

Printing Support

Assembly Monitoring—This functionality allows the user to monitor theassembly of tissue constructs using a mounted camera located within theprinter. Once this option is enabled, a video feed appears in TSIM thatstreams until the printing process is finished.

Digital Prototyping—Simulation Tools—A suite of simulation tools areavailable within the Simulation module of TSIM. There is a tool forestimated print time (Print Time tool), required volume for extrusion(Volume tool), and structural analysis of the 3-D model (StructuralAnalysis tool).

Print Time Tool—This tool estimates and displays the time (in minutes)that it will take to print the created 3-D model.

Volume Tool—This tool estimates and displays the volume of extrusionmaterial that is required to print the user's 3D model.

Structural Analysis Tool—This tool allows the user to perform astructural analysis assessment on the 3-D model to estimate itsstability once printed. This tool is based on the principles of smoothparticle hydrodynamics.

Material Database MD

A materials database MD is available and defines what extrusionmaterials are available to the user when modeling tissue constructs.Users are able to find their desired materials by searching all of theavailable materials, browsing all of the available materials, orsearching available materials by application. This materials database MDpermits users to add or edit entries, with delete functionality reservedto the Local Database of the user. In addition to pictures of thematerials, information stored for the materials will include parameterssuch as material type, viscosity, weight percent (in solution), Young'smodulus, stress, and strain.

Local Database, A local database comprises a basic materials databaseincluding pre-set materials. The user may then expand upon this databaseand save new entries on a local machine.

A specific fabrication material may be assigned to each object, and eachmaterial is associated with a dispensing needle type (length, diameter)and operational parameters including pressure, speed, acceleration,on/off delay and cure methods, Once assigned, a material selection andmaterial details may be edited in accordance with test print feedbackand/or specific application needs. In the alternative, a user may createa custom materials menu adapted to specific application needs.

Once added to the modeling screen, an object may be transformed. Inspecific embodiments, a transform function selection panel is provided.In very specific embodiments the object may be transformed by moving theobject and/or modifying the center coordinates of the object. Otherexamples of transforming functionality include rotating an object aboutits center coordinates, for example by dragging individual roll, pitchor yaw sliders or by dragging an individual block in a desireddirection, and scaling an object, for example by scaling via a uniformxyz scale factor or by scaling along an individual axis. In veryspecific embodiments transforming functionalities are reversible.

Once designed, models to be printed/fabricated are sent to the RIM/usingthe TSIM software. The RBW comprises a tabletop workstation including amulti-axis robot that facilitates 3-D tissue printing of compositevolumetric shapes and assembly of biological constructs. The robotcomprises at least six axes of fabrication orientation making itparticularly suitable for the level of precision required to assemblefunctional tissue structures. According to one embodiment, the RBWcomprises a multi-axis robot, a robotic controller (in preferredembodiments the controller is located outside the RBW housing or isseparately contained within the RBW housing), a frame defining ahousing, a print stage, at least one robot end effector, at least onematerial storage unit, and a unit interface (in preferred embodimentsthe RBW-UI is located outside the RBW housing). In specific embodimentsthe robot comprises an arm having at least a six-axis range of motion.In very specific embodiments the robot is selected from an EPSON C3series compact 6-axis robot compatible with a PC based con roller and orthe EPSON RC180 and RC620+ PC based controllers. A frame may compriseone or more of the following features: a Bosch aluminum T-slot, doors,floor, ceiling, roof, paneling, and lighting. A print stage may beconstructed of any suitable material and in specific embodimentscomprises a leveling mechanism. In preferred embodiments, the robot issituated over and above the print stage. A Robot end effector comprisesone or more of a pneumatic gripper, syringe barrel holder, camera, laserdisplacement sensor, auto-leveling chuck, and cable management. Inspecific embodiments a material storage unit comprises a syringe holder,displacement sensors and a station mount. The electrical design includesa pneumatic feed line and an electrical power cable. The workstation maybe provided in mobile form, for example on a cart.

The RBW User Interface (UI) comprises a status screen which provides theuser with information relating to the current state of the workstation.From the status screen a user may, for example, monitor the properfunctioning of the RBW's components, such as the controller, dispenserand microcontroller, view a streaming video feed of print jobs, andfollow-up on any alarms indicating attention required before proceeding.

The RBW-UI further permits a user to make offsets, which is a minormodification made in real-time based on empirical observation. Offsetsmay be made for pressure, vacuum, speed and Z-height and accelerationvalues, for example.

Features of the robotic bioassembly workstation are described now withreference to specific embodiments depicted in FIGS. 1, 2 and 3 . Itshould be noted that this discussion is for illustrative purposes, and aperson of ordinary skill in the art will appreciate that variousconfigurations are possible without departing from the scope of theinvention as defined by the claims. As depicted in FIG. 1 , an RBWcomprises a framed housing 1 comprising a multi-axis robot 2 comprisinga robotic arm 3 having a robotic are effector component 4, a roboticcontroller 5, a material storage unit 6, a material dispensing system 7,an adjustable print stage 8, and a user interface 9. In preferredembodiments the framed housing is operationally accessible to a userfrom multiple angles and provides at least one real-time observationaccess. The RBW may also be fitted with a camera 10 as part of anintelligent visual feedback system to supplement empirical observationand/or to provide remote viewing options, as well as the ability to stopa print/assembly process and make adjustments.

The bioassembly system comprises a multi-axis robot 2 and in preferredembodiments is a six-axis robot. In specific embodiments the materialdispensing system 7 comprises extrusion syringe dispensers 11 adaptedfor direct-writing a biomaterial 12 onto a substrate. Due to theincreased degree of freedom of the six-axis system, dispensing ofbiomaterial may be effectuated at an angle oblique to a plane of asurface of a printing substrate.

In specific embodiments, the robotic arm effector component 4 comprisesone or more effectors selected from printing tools, staging andassembling tools, and sensors, and the system further comprisesautomatic tool exchange functionality for effectuating automatedexchange of tools at the effector component as dictated by the printand/or assembly command. According to specific embodiments, printingtools are selected from a gripper, a syringe barrel 13 adapting holder14 and a dispenser H. Staging and assembling tools are selected frompicking, placing, and positioning tools, and sensors are selected from alaser displacement sensor and a photoelectric sensor. In certainembodiments the RBW comprises a print stage 8 leveling mechanism. Inspecific embodiments, a laser displacement sensor is located on therobotic arm effector 4. The laser displacement sensor enhances a manualleveling protocol.

Staging in accordance with certain embodiments may include picking andplacing a print substrate onto the print stage. Printing is theneffectuated on the substrate, which in very specific embodiments mayinclude variable surface topographies designed according to needs ofspecialized constructs, Assembling an ultimate bioconstruct may beeffectuated by, for example, picking and positioning a first printedconstruct relative to a second construct, the second construct selectedfrom a second printed construct and a provided construct. The printstage 8 may be partitioned into areas, for example a print area and anassembly area, and non biological constructs may be included forassembly with biological constructs. Medical devices and jigs may becomprised entirely of non-biological materials; however fabrication mayproceed similarly and advantages conferred by the bioassembly system forthe fabrication of biological constructs also generally apply tonon-biological constructs.

Generally, the material dispensing system 7 comprises multiple syringes11, each syringe containing one material or bio-material 12, anddispensing from a syringe 11 comprises dispensing one material orbiomaterial 12 at a time. The material storage unit 6 comprises at leastone syringe barrel holder 14. Each syringe barrel holder 14 comprisesmultiple syringe barrels 13. The material storage unit 6 may alsoinclude at least one displacement sensor for ensuring correct seating ofsyringe barrels 13 in the holder 14; and at least one needle detectionsensor for detecting needle 16 size and tip deflection.

One embodiment is directed to a robotic biomaterial dispensing apparatuscomprising a robotic arm and a robotic arm end effector, the endeffector configured to grip and secure a dispensing syringe, wherein therobotic arm provides movement of the syringe along at least six axes.The ability to move along six axes permits novel functionality, inparticular with respect to printing on 3-D print substrates.Bio-constructs may be fabricated, for example, by dispensing biomaterialonto a print substrate without being restricted to sequential layeringas with conventional bioprinter designs, which rely on fabricatingcomplex constructs such as tissues and organs layer by layer. With sixaxes, biomaterials may be dispensed by non-sequential planar layeringsuch that the robot effector may return to a prior layer and add morebiomaterial after dispensing a subsequent layer. Further, the roboticeffector is capable of aligning a dispenser/syringe tip at an anglenormal to any point on a contoured surface. This provides a higherdegree of 3-D printing versatility and permits a wider variety ofsurface contours to completed constructs, and with respect to objectsderived from medical imaging, provides potential for construction from aradial geometry, utilization of polar or hybrid polar coordinatesystems, and therefore greater 3-D precision. Specific embodiments aredirected to methods of fabricating a 3-D biological construct comprisingdirect-write dispensing of one or more biomaterials onto a 3-D printsubstrate utilizing the robotic biomaterial apparatus according to claim24, wherein the robotic arm end effector positions the dispensingsyringe at an angle normal to all positions on a surface of the 3-Dprint substrate.

EXAMPLES

The Following Examples are set forth to illustrate certain aspects,features and advantages of embodiments of the instant invention andshould not be construed as limiting the scope of the invention asdefined by the appended claims.

Example 1. This example illustrates creation of simple objects in theobject modeling environment.

A. Create a box (FIG. 7 )

-   -   1. Click on the Create a Box icon.    -   2. Click the location on the modeling environment to begin        placing the Box.    -   3. The size of the Box's base can be adjusted by moving the        mouse accordingly. To accept, click the left-mouse button to set        the base.    -   4. Moving the mouse vertically sets the height of the Box, To        accept, click to set the height. Further refinements to the        dimensions of the Box can be made by manually editing the        center's coordinates or changing the length/depth/height of the        Box.        B. Create a Sphere in the modeling environment (FIG. 8 ),    -   1. Click on the Create a Sphere icon.    -   2. Click the location in the modeling environment on which to        place the sphere.    -   3. The size of the sphere can be adjusted by moving the mouse.        To set the size, click once more. The sphere may be further        refined by manually entering the center's coordinates or        changing the value of the radius.    -   4. To accept the final shape, click the Create Solid button on        the Sphere menu.        C. Create a Cylinder in the modeling environment (FIG. 9 )    -   1. Click on the Create a Cylinder icon.    -   2. Click the location in the modeling environment on which to        place the cylinder.    -   3. The size of the cylinder's base can be adjusted by moving the        mouse. To accept, click to set the base.    -   4. Next, adjust the height of the cylinder by moving the mouse        and clicking once more to set it.        -   Dimensions of the cylinder may be further refined by            manually entering the center's coordinates or the value of            the radius or the height.    -   5. To accept the final shape, click the Create Solid.        D. Create a Pyramid in the modeling environment (FIG. 10 )    -   1. Click on the Pyramid Tool icon.    -   2. Click the location in the modeling environment on which to        place the pyramid.    -   3. The size of the pyramid's base can be adjusted by moving the        mouse. To accept, click to set the base.    -   4. Next, set the height of the pyramid by moving the mouse. To        accept, click to set the model.    -   5. The dimensions of the pyramid can be further refined by        manually entering the center coordinates or the value of the        radius or height.    -   6. To accept the final shape, click the Create Solid.

Example 2. This Example illustrates manipulation of the objects in theobject modeling environment. The objects added to the object modelingenvironment may be further manipulated, for example by combining, movingor aligning objects. In particular embodiments, objects may be combinedto form unique/customized shapes. Operations for combining may includeadding (union), subtracting (difference) and/or intersecting objects inthe modeling environment.

A Union/Combine operation comprises merging together two separate objectvolumes so that their boundaries will be defined as a single region. Aunion operation is illustrated in FIG. 11 .

A different operation is used to define a region boundary based on thesubtraction of an actively selected object from an inactive object. Adifference operation is illustrated in FIG. 12 .

An intersection operation is used to define a region boundary based onthe intersection of two objects. An intersection operation isillustrated in FIG. 13 .

Functionality for aligning objects in the modeling environment is alsoprovided. In specific embodiments, two faces on different objects may bealigned based on a point selected on each shape, as illustrated in FIG.14 .

In other specific embodiments, objects may be aligned along the y-axis,x-axis or z-axis based on center points of selected facades/sides, Amove operation permits movement of objects into different positionswithout necessarily aligning (FIG. 15A).

A dragging operation, as illustrated in FIG. 15B, permits moving aselected shape in different directions relative to the “ground”, whichis determined based on a camera angle. In one specific example, in adefault view, dragging a model will move a shape in the xy-plane.

A measuring operation permitting point to point measurement lines onboth the 2-D image slices and within a 3-D model is also provided andillustrated in FIG. 15B.

Example 3. This example illustrates importing objects directly into themodeling environment from other programs. Users may open existing modelfiles created in external programs. Exemplary importable file formatsinclude STL, NIfTI and DICOM.

Importing an STL file,

-   -   1. Click on the Import STL icon in the Import section.    -   2. Navigate to the folder containing the STL files.    -   3. Highlight the desired file and click the ‘Open’ button to        import the model.    -   4. The object will appear in the modeling environment. As in the        case of shapes created in TSIM, modification of the imported        object is via the Objects menu on the right-hand side.        B. Importing a NIfTI file.    -   1. Click on the Import NIfTI icon in the Import section.    -   2. Navigate to the folder containing the NIfTI files    -   3. Highlight the desired file and click the ‘Open’ button to        import the model    -   4. The object will appear in the modeling environment. As in the        case of shapes created in TSIM, modification of the imported        object is via the Objects menu on the right-hand side.        C. Importing a DICOM file.        An example of an imported WON image is depicted in FIG. 16 .    -   1. Click on the DICOM icon in the Import section.    -   2. Navigate to the folder containing the DICOM files.    -   3. Click ‘Select Folder’ to import the model.

Example 4. This Example, taken with FIG. 16 , illustrates a volumeoperation, which permits further analysis of a volumetric model (e.g. NMor DICOM) by adjusting density or opacity values. The spacing values foreach axis may be modified as well.

-   -   Import the volumetric file    -   Click on the Volume icon    -   Click on the imported model to highlight it. The Adjust Volume        menu will now be displayed on the right hand side. By adjusting        the individual sliders, you can change the respective value,        which in turn updates the model. The viewing angle may be        changed while making adjustments to the volume.

Example 5. This example illustrates a sketch operation according tospecific embodiments.

-   -   1. Click on the Create a sketch icon    -   2. The three individual planes will appear in yellow. To choose        the plane onto which a sketch will be drawn, click on a desired        plane.    -   3. The chosen plane will now appear highlighted in blue together        with the interaction widgets. The plane may be moved or rotated.    -   4. Click on the Create button to begin working on a sketch.    -   5. If selection of a different plane is desired, choose the        Select a New Plane button to return to Step 2.    -   6. The selected plane will now be displayed as a grid and the        Sketch menu will appear. Custom shapes may be drawn by using        curves, circles, points, rectangles, etc.    -   7. Once a shape drawing is completed, right-click to stop        drawing and click on the Select tool to return back to the Solid        Modeling screen.

Sketches may also be modified and managed.

More complicated shapes may comprise a collection of sketches made ondifferent planes. Managing operations permit users to modify, move,delete or hide individual sketches in a collection. Sketches may beinteracted with individually via the sketches menu.

Example 6. This example illustrates extrusion of a sketch into a 3-Dmodel (FIG. 17 ).

-   -   1. Begin with an overhead view of the sketch.    -   2. Next, hold the CTRL key and click on each of the individual        sides of the sketch. Selected sides will be highlighted in blue.    -   3. A wire frame of the shape is displayed when all sides are        connected. The Extrude menu on the right side will now be active        and allows adjustment of both the length and resolution of the        extrusion.    -   4. Click on the Extrude button to create the final shape.

Example 7. As shown in FIG. 18 , new shapes may be created by connectingboundaries of sketches rendered on different planes, with furthermodification possible by lofting contours.

Example 8. This example illustrates utilization of the TSIMfunctionality to model a vascular tree (FIG. 20 ), which may then befabricated via the RBW.

According to one specific embodiment, the method comprises entering in aset of input parameters such as those set forth in Table 1.

TABLE 1 Starting diameter (μm) Diameter of the largest vessel at theinitiation end. Order length (μm) length of an order. Ratio Determinesthe decrease in size by ratio of diameter from one order to the next.Number of orders The number of steps down in the branching patternEnding diameter (μm) Diameter of the largest vessel at the terminal end.

Once a model is generated, specific materials may be assigned to eachorder of the vascular tree. In specific embodiments, the RBW stages theprint stage with a mold or other substrate having a variable surfacetopography on which to print a vascular tree. Printing of other complextissue constructs may also require staging by picking and positioningsubstrates as printing surfaces into the print stage. In some specificembodiments, a print stage may be divided into a print area and anassembly area so that a bin-construct may be assembled as it is printedon a component by component basis.

Example 9. This example illustrates modeling of a 3-D tube from asketched curve, further as depicted in FIG. 19 . Radius and resolutionof a generated tube may be directed and modified.

Example 10. This example, taken with FIG. 21 , illustrates initiation ofprinting by the robotic bioassembly workstation.

-   -   Print job is chosen from the list. A preview is available under        the Project Preview,    -   Press Start    -   After a successful password prompt, a material loading process        can begin.

In very specific embodiments, once a print job has been chosen andstarted, the UI will prompt the user to physically load the individualbarrels into specific slots on the material storage unit. Once loaded,the user confirms before moving on to the next material. Visualindicators may be present to provide a status update of each of thematerial holder bays. Table 2 is illustrative of this concept.

TABLE 2 Color Meaning Grey The specific bay is occupied with a barrel.Green Load the material in this bay Red Incorrect material placed in thebay.

A “Move” operation permits manual adjustment of positioning of the robotto place it into a desired position via the RBW-UI screen. The robot maybe adjusted along any axis and the robot may be restored to a “home”position. Other control operations may include resetting, power to acontroller, adjusting and turning on the pressure (psi), turning offindividual joint brakes (J1 through J6, one J for each movement axis). Acalibration menu ensures that the workstation is setup accordingly aheadof any prints made.

Example 11. This Example illustrates an embodiment of a materialstesting protocol prior to printing/fabrication. When utilizing amaterial for the first time or under unique condition parameters, it isimportant to test printing quality at various needle diameters, speeds,pressure, etc.

Exemplary steps to conducting a material test include:

-   -   1) Manually attach a syringe with a new material to the robot's        end effector.    -   2) On the RBW-UI, press the Material Test option under        Calibration.    -   3) grid of the printing platform will be displayed, To begin,        press one of the cells in the grid to select it.    -   4) Increment or decrement the desired parameter value for that        cell by pressing the respective plus or minus sign.        Alternatively, click in the value box for a parameter and use        the on-screen keyboard to enter desired values.    -   5) Press Save to set the values for the selected cell.    -   6) If desired, choose another cell and repeat steps 4-5.    -   7) When ready to print, press Start. Please note, the Start        command does not appear until values for at least one cell are        saved.

A completed print will be lines printed with the material and it can bedetermined which group of settings best meets specific applicationneeds.

Example 12. This example illustrates print stage/platform levelingfunctionality.

A level print stage is intrinsic to producing quality prints. Inspecific embodiments, the RBW-UI provides feedback relating to thedegree to which the stage is level and what modifications may need to bemade to perfect leveling.

To begin leveling:

-   -   1) Press the Leveling option located under Calibration.    -   2) Press Start to begin the leveling test.    -   3) As the robot moves to a position near each stage bolt, the        height of the print platform is determined. At the conclusion of        the test, a user is prompted with the exact adjustment to raise        or lower the individual corners.

The distance from the top of the robotic arm to the center of the stagemust also be calibrated. A tip sensor is detected and position isdetermined. An offset of barrel to sensor is determined and a pressuredisplacement curve is calculated.

Example 13. This example illustrates exemplary components andcomponent-associated functionality of a very specific embodiment of theRBW according to the disclosure. Reference is to elements designated inFIGS. 1, 2 and 3 .

Housing

Frame: According to one non-limiting exemplary embodiment the frame isan aluminum T-slot Frame. For example, the RBW physical frame may beconstructed using a combination of Bosch T-Slot Profiles,

Doors: The doors of the RBW are constructed using Bosch T-Slot Profileswith clear, scratch-resistant polycarbonate inserts to provide userswith visibility of the inside of the station.

Floor: The RBW floor may be a solid ⅛″ thick aluminum sheet with threeholes for mounting and adjusting the print stage.

Ceiling: The RBW ceiling may be a solid thick aluminum sheet.

Roof: The RBW roof may be a solid ¼″ thick dove-grey acrylic sheet.

Fascia Paneling: The fascia paneling of the RBW can be constructed using¼″ thick dove-grey acrylic sheets. In preferred embodiments, panelsutilize M5 black knurled head thumb screws to fasten to the frame.Panels are removable to allow users access to the station control panel.

Lighting: The RBW may have four white LED lights (IP68 rated) mounted inthe four corners of the station to enable a camera on the robot endeffector to achieve a desired resolution.

2. Print Stage

Material of construction: The RBW print stage may be comprised of asolid sheet of acrylic. It may comprise three counterbore holes forrecessing the bolt heads of the stage leveling bolts. In very specificembodiments, a leveling the bolts may be effectuated manually with aratchet wrench.

Leveling mechanism L1: According to specific embodiments, the RBW printstage comprises three leveling bolts that insert into the through-holesof the three counterbore holes of the stage and extend down into threetapped mounting holes located in the channels of the support profilesbelow the floor of the station. A spring is wrapped around each boltin-between the print stage and station floor. The diameter of thecounterbore hole is such that a tool can be inserted to tighten orloosen the stage bolts.

Robot End Effector:

The robot end of arm tooling, herein designated as an “end effector,”comprises components that attach to J5 and J6 (of the 6-axis robot) andincludes print tools, assembly tools and sensors. Print tools include,e.g. a syringe barrel adapter holder and gripper.

Pneumatic gripper: According to specific embodiments, the robotic endeffector comprises a pneumatic gripper mounted on its end, with grippingfingers oriented perpendicular to the centerline of the robot's J6 axisof rotation. The air feed to the gripper may be controlled by a solenoidvalve that is turned on and off by the RBW control software. The gripperis used to grip one material syringe barrel 13 at a time for the purposeof loading and unloading the barrels 13 from the materials storing unit6.

Syringe barrel holder 14: In very specific embodiments, a second pieceof the RBW end effector connects a dispenser to syringe barrels 13, forexample a Norson EFD Ultimus V dispenser to Nordson EFD 30 syringebarrels using an adapter assembly from Nordson EFD. This piece housesthe adapter assembly from Nordson EFD on the end of axis J6 of therobot, allowing syringe barrels 13 to be screwed into the assembly andextrusion to be controlled using Ultimus V dispensers.

Automatic tool change: According to particular embodiments, the RBW iscapable of auto-changing from print/dispensing tools to specificallydesigned end-effector tools for pick and place of receptacles such asmolds or containers to facilitate specific print applications. The autotool change capability also enables the selection of tooling from thetool holder and used for assembling individual printed structures intomore complex structures.

Camera: This element allows the user to monitor the printing of tissueconstructs using a mounted camera located within the housing. Once thisoption is enabled, a video feed appears in TSIM and at the RBW userinterface (RBW-UI) that streams until the printing process is finished.

Laser displacement sensor S1: In some embodiments, a laser displacementsensor S1 is mounted on the end effector in parallel with the pneumaticgripper and syringe barrel adapter holder. This laser sensor is used tomeasure the distance between the print stage and end effector during thestage leveling process.

Photoelectric Sensor: A photoelectric sensor may be mounted on the endeffector which allows the robot to detect placement of barrel holderswhen it is near. In addition, the sensor detects that the barrels arecorrectly picked up from the respective holder.

4. Material Storage Unit 6

Syringe holder 14: According to a specific illustrative embodiment, RBWhouses four syringe holders 14 and each holder 14 contains threematerial cartridges 13 for a total of 12 syringes 13.

Displacement sensors S1: The through beam sensor S1 runs along the topof the material storage unit and checks to ensure the correct seating ofthe syringe barrels. In this exemplary embodiment there are eightthrough beam sensors S1 located on the front and back side of each ofthe barrel holders. A stand-alone sensor (needle detection sensor S2),located at the bottom of the material storage unit, is used to detectthe size of the needles and needle tip deflection for each of thesyringe barrels.

Computer/Touchscreen (RBW-UI)

Location: In certain embodiments, the RBW-UI is comprised of atouchscreen computer mounted on a swivel arm located on an exterior partof the physical frame of the workstation.

Interface: The RBW-UI provides information about the printer's currentstate regarding the parameters of pressure and speed. A chart showingthe amount of material used by the printer is displayed on the UI. Theuser is notified upon completion of print job, Dialog boxes pop up if anerror is encountered in the assembly/bioassembly process. For example,if the RBW runs out of material in a cartridge, a dialog box will pop upon screen that says, “Fill Reservoir 2 with Gel 2.”

The interface's Status option allows the user to check the status of theRBW by viewing the existing print jobs, a history, and a video feed ofthe assembly/bioassembly stage. The interface's Direct Control optionprovides the ability to job the robot to a desired location via thetouchscreen.

In exemplary embodiments, the Leveling option may have the robot checkone position near each stage leveling bolt and determines how level theprint stage is using values obtained from the laser displacement sensorat each position. The Offsets option allows the user to manually setoffsets. The Materials Testing option allows for the testing ofmaterials with different parameters.

Controls Designs

Safety: Safety features comprise an emergency stop to shut down therobot entirely. Additionally, door locks may be included an added safetymechanism. Door locks halt operation in the event the door is openedduring fabrication.

Controls container: The controls container is securely mounted onto theRBW in the overhead controls area and serves as housing for themicrocontroller.

Layout of overhead controls area: The overhead controls area consists ofthe controller, for example an RC180 controller, the controls container,the Nordson EFT Ultimus V dispenser, a 220 v step up transformer andassociated components.

Microcontroller: Together with the printed circuit board (PCB) daughterboard, the microcontroller interfaces between the and the IO monitoring.

Printed Circuit Board (PCB)

7. Electrical Design

Pneumatic feed line: According to very specific embodiments, a singlepneumatic quick connect receptacle is located on the right side of theRBW. Users connect input air line here. This line runs internallythrough the RBW to a regulator that then splits and feeds the line tothe dispenser and solenoid valve.

In an illustrative embodiment, three pneumatic feed lines may then berouted to the robot. Two lines are used by the gripper. The remainingfeed line is used to depress the barrel piston for dispensing.

Electrical Power Cable

8. Accessories

Mobile cart: in some embodiments, the RBW is housed on a mobileinstrument cart. In very specific exemplary embodiment, the dimensionsof the cart include two steel shelves 30″×40″ with 1½″ retaining lips,the overall cart height is 29″ with a 27½″ top shelf surface height andthere are two swivel and two rigid 8″×2″ phenolic wheels with wheel lockbrakes on the swivel casters.

What is claimed:
 1. A robotic biomaterial dispensing apparatuscomprising: a robotic arm and a robotic arm end effector, the endeffector configured to grip and secure a syringe barrel, wherein therobotic arm provides movement of a syringe along at least six axes; amaterial storage and dispensing system comprising a material storageunit comprising at least one syringe barrel holder, each barrel holdercomprising multiple syringes, and the material storage unit furthercomprising at least one needle detection sensor for detecting needlesize and tip deflection; and an adjustable print stage comprising aleveling mechanism configured to measure a height of the adjustableprint stage and output a height adjustment recommendation, wherein theheight of adjustable print stage is configured to be adjusted based onthe height adjustment recommendation from the leveling mechanism,wherein the robotic arm is configured to print biomaterial from thesyringe from a plurality of directions onto a surface of the adjustableprint stage based on an adjustable movement of the robotic arm andwherein the apparatus is contained within a housing.
 2. A method offabricating a 3-D biological construct comprising utilizing the roboticbiomaterial dispensing apparatus according to claim 1 to direct-writedispense one or more biomaterials onto a print substrate bynon-sequential planar layering.
 3. A method of fabricating a 3-Dbiological construct comprising utilizing the robotic biomaterialdispensing apparatus according to claim 1 to direct-write dispense oneor more biomaterials onto a 3-D print substrate, wherein the robotic armend effector positions the dispensing syringe at an angle normal to twoor more desired surface positions of the 3-D print substrate.
 4. Arobotic material dispensing apparatus comprising: a robotic arm and arobotic arm end effector, the robotic arm end effector configured togrip and secure a cartridge comprising a syringe barrel from a materialstorage unit, wherein the robotic arm provides movement of the cartridgealong at least six axes; the material storage unit comprising at leastone syringe barrel holder, each syringe barrel holder comprisingmultiple syringe barrels and at least one displacement sensor forensuring correct seating of syringe barrels in the holder, wherein thematerial storage unit further comprises an at least one needle detectionsensor for detecting needle size and tip deflection; and an adjustableprint stage configured to be automatically adjusted and comprising asurface to receive a printed material, wherein the robotic arm endeffector comprises a laser displacement sensor and the adjustable printstage comprising a leveling mechanism enhanced by feedback from thelaser displacement sensor, the leveling mechanism configured to measurea height of the adjustable print stage and output a height adjustmentrecommendation, wherein the height of adjustable print stage isconfigured to be adjusted based on the height adjustment recommendationfrom the leveling mechanism.
 5. A method of fabricating a 3-D constructcomprising utilizing the robotic material dispensing apparatus accordingto claim 4 to direct-write dispense one or more materials onto a printsubstrate by non-sequential planar layering.
 6. A method of fabricatinga 3-D construct comprising utilizing the robotic material dispensingapparatus according to claim 4 to direct-write dispense one or morematerials onto a 3-D print substrate, wherein the robotic arm endeffector positions the cartridge at an angle normal to two or moredesired surface positions of the 3-D print substrate.
 7. A 3-D design,fabrication and assembly system comprising: a modeling component and arobotic assembly workstation component, a hardware processor coupled toa memory, the modeling component comprising a user interface, at leastone suite of tools for performing an object operation selected fromcreating, editing, modeling, transforming, image property modulating,sketching, print supporting, simulating, material testing andcombinations thereof, a material database, and software executable bythe hardware processor and configured to cause the hardware processor,when executed by the hardware processor, to facilitate a method fordesigning a volumetric model of a construct at the user interface, themodeling component being operationally linked to the robotic assemblyworkstation component, the robotic assembly workstation componentcomprising a framed housing comprising a multi-axis robot comprising arobotic arm having a robotic arm effector component, a roboticcontroller, a material storage unit, a material dispensing system, andan adjustable print stage, wherein the robotic arm effector componentcomprises a laser displacement sensor and the adjustable print stagecomprises a leveling mechanism enhanced by feedback from the laserdisplacement sensor, the leveling mechanism configured to measure aheight of the adjustable print stage and output a height adjustmentrecommendation, wherein the height of adjustable print stage isconfigured to be adjusted based on the height adjustment recommendationfrom the leveling mechanism, and wherein the robotic arm is configuredto print material from a plurality of directions onto a surface of theadjustable print stage based on an adjustable movement of the roboticarm.